A. Superoxide Dismutase
Superoxide radicals and other highly reactive oxygen species are harmful by-products produced in every respiring cell, causing oxidative damage to a wide variety of macromolecules and cellular components. A group of metalloproteins known as superoxide dismutases (SOD) catalyzes the oxidation-reduction reaction 202−+2H+→H2O2+O2 and thus provides a defense mechanism against oxygen toxicity. SOD may contain manganese or iron or a combination of copper and zinc (U.S. Pat. No. 5,540,911).
Oxidative damage has been positively correlated with a reduction in MnSOD activity (Isoherranen et al., J Photobiol (1997) 40(3):288–293); hence, SODs from various sources are currently of great interest as potential therapeutic treatments for oxidative damage. Their use in a clinical setting for the treatment of a wide variety of disorders has been proposed (see Beck et al., Nuc Acids Res (1988) 15(21): 9076). These proposals include (i) prevention of oncogenesis, tumor promotion and invasiveness, and UV-induced damage; (ii) protection of cardiac tissue against post-ischemia reperfusion damage; (iii) as an anti-inflammatory agent; (iv) to reduce the cytotoxic and cardiotoxic effects of anticancer drugs, and; (v) to improve the longevity of living cells (U.S. Pat. No. 5,772,996).
The importance of reactive oxygen species (ROS) or changes in the cellular redox state in the pathogenesis of viral infections is becoming increasingly evident. For example, influenza virus infection of the lungs induces MnSOD expression and thus is associated with oxidative stress (Choi et al., Am J. Physiology (1996) 271(3 Pt 1): L383–391). Further, SOD, specifically MnSOD, is recognized in the art to be modulated during various stages of viral infection (e.g., Choi et al Am J. Physiology (1996) 271(3 Pt. 1): L383–391; Ritter et al., J Exp Med (1994) 185(5): 1995–1998; Jacoby et al., Free Radic Biol Med (1994) 16(6): 821–824; and Carbonari et al., (1997) 90(1): 209–216). This is especially acute in HIV infections, wherein the effect of cytokines, such as TNF alpha, may further exacerbate the effects of oxidative stress in the infective pathogenesis associated with AIDS (Le Naour, et al., Res Immunol (1992) 143(1): 49–56 and Shatrov et al., Eur Cytokine Network (1997) 8(1): 37–43).
Patients infected with HIV-1 often display destroyed immune cells in the peripheral lymphoid tissues as well as exhibit cognitive defects that are related to progressive neuronal degeneration and cell death involving the redox state (New et al., Neurovirol (1997) 3(2): 168–173 and Flores et al., Proc Natl Acad Sci USA (1993) 90(16): 7632–7636). HIV-infected patients have also shown low levels of MnSOD (Zhang et al., Antioxidants and AIDS, 36–37 (CRC Press LLC, 1997). Apoptosis and aging involve oxidative stress as measured by degradation of mtDNA (Ozawa T., Biosci Rep (1997) 17(3):237–250). What is of critical relevance is that all of these events reflect the involvement of moderate to severe mitochondrial damage (e.g., mtDNA deletions) in part due to the effects of ROS (Kniman et al., Exp Neurol (1998) 154(2): 276–288; and Carbonari et al., (1997) 90(1): 209–216).
B. Mitochondrial Damage
To meet the body's acute and chronic energy demands, all cells of the body synthesize their fuel within their mitochondria. Mitochondrial DNA (mtDNA) contains thirteen genes encoding protein products that are necessary to synthesize the cell's fuel, adenosine triphosphate (ATP). All cells have mitochondria, but the number of mitochondria per cell can vary from a few hundred to tens of thousands per cell.
Mitochondrial oxidative stress has often been implicated as an initiator of the mtDNA mutation process because mitochondria consume most of the cell's oxygen for ATP synthesis. If oxygen is not metabolized efficiently, oxygen free radicals can accumulate in the cell. Free radicals can cause protein and lipid peroxidation, as well as oxidative damage to the mtDNA. If oxidative damage to mtDNA is left unrepaired, the point-mutation and deletion rate of the mtDNA increases, which may eventually lead to permanent organ fatigue.
A random mutation on one mtDNA molecule would not alone be deleterious to an organism due to the large intracellular pool of mtDNA. Accordingly, a few random mtDNA mutations may have no distinguishable phenotypic effects, but once a high level of these mtDNA mutations has accumulated in critical cells, an energy-loss syndrome may occur which could effect the heart (cardiomyopathy or conduction disorders), the brain (seizures, dementia), pancreas (non-insulin dependent diabetes), the gastrointestinal tract (dysmotility or pseudo-obstruction), inner ear (sensorineural hearing loss), kidney (glomerulopathy) and/or the skeletal muscle (myopathy).
As oxidative stress can induce both mtDNA mutations and apoptotic death, the discovery that mtDNA mutations are the basis of a number of human pathologies may have profound implications.
C. Cell Death
Programmed cell death (sometimes referred to as apoptosis) is distinguishable, both morphologically and functionally, from necrosis. Programmed cell death is a natural cellular event. Cells dying by programmed cell death usually shrink, rarely lyse, and are efficiently removed from the organism without the appearance of inflammation. Necrosis, however, is a pathological type of cell death observed following physical or chemical injury, exposure to toxins or ischemia (lack of oxygen). Dead cells are rapidly recognized and engulfed by macrophages (Michael Hengartner, Cell Death and Aging, Molecular Mechanisms of, MOLECULAR BIOLOGY AND BIOTECHNOLOGY, 158–62 (ed. R. A. Meyers, 1995)).
It is becoming increasingly clear that oxygen metabolism plays a key control point in programmed cell death or apoptosis. Therefore, identifying the gene product responsible for apoptotic oxidative stress is key to therapeutic drug development. In cancer, for example, it would be therapeutically advantageous to be able to induce apoptosis in malignant cells by increasing oxidative stress in these cells.
Conversely, inhibiting oxidative stress may prolong cell life. The immune activation of T cells of HIV-infected individuals leads to oxidative damage of proteins and lipids and apoptotic T cell death (Piedimonte et al. (1997) Infect Dis 176: 655–664; Walmsley et al. (1997) AIDS 11 (14):1689–1697; Groux et al. (1992) J Exp Med 175: 331–340). Oxidative stress also causes HIV viral load to increase (Schreck et al. EMBO J (1991) 10 (8):2247–2258; Staal et al., Proc Natl Acad Sci USA (1990) 87: 9943–9947). Therefore, inhibiting oxidative stress might be beneficial in preventing T cell depletion in HIV-infected individuals.
Therefore, given the importance of the redox state on cell function and homeostasis, there is a need to identify any gene and/or gene product having a role in oxidative stress.